Dihydro and hexahydro isoalpha acids having a high ratio of trans to cis isomers, production thereof, and products containing the same

ABSTRACT

This invention describes heretofore unknown forms of dihydro (DHIA) and hexahydro (HHIA) isoalpha acids having a high ratio of trans to cis isomers and a process for their production. Also, non-precipitating clear 5, 10, 20% and higher aqueous solutions thereof, since they are soluble at room temperature in soft water. This is due to the high ratio of trans to cis isomers. Unlike prior art essentially all cis isomer products, they remain haze free both at a neutral pH in water and at 1% to 2% and higher concentrations. This invention has the advantage over the prior art in that DHIA and HHIA can be provided as stable, non-separating liquids, at practical concentrations in the range of 5% to about 40%, which do not require heating to about 50° to 90° C. and above with stirring to effect dissolution of precipitates. The high trans products described herein can be admixed with isoalpha- and tetrahydro-isoalpha acids.

The present application is a division of application Ser. No. 09/512,944of Feb. 25, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Dihydro and hexahydro isoalpha acids having a high ratio of trans to cisisomers, process for the production thereof, and products containing thesame.

2. Prior Art

There are four types of isoalpha acids: the unreduced form, calledisoalpha acids (isohumulone) (IA), and three types of reduced forms ofIA. The latter are dihydro-isoalpha acids (DHIA), also known as “rho”,tetrahydro-isoalpha acids (THIA), and hexahydro-isoalpha acids (HHIA).Each is present as three major analogues differing in an acyl side chain(the co, n, and ad analogues) and as trans and cis and optical isomers.The proportions of analogues depends upon the variety of hops used tomake the iso acids. Only IA, DHIA, and THIA have been and are availableas aqueous forms. Their structures are shown in FIGS. 1 and 2.

IA and THIA do not form insoluble crystalline precipitates uponstanding, due to their chemical composition, which includes a keto groupon the lower acyl side chain. Commercially available all cis isomer DHIAand HHIA have this keto group reduced to an alcohol. They formprecipitates over time, which are exceptionally hard to redissolve.Their solubility in water at pH 10 is about 1%, and much less at pH 7 to8. The products described herein, containing large amounts of the transisomers of DHIA and HHIA, are remarkably and unexpectedly soluble inwater and overcome this limitation, being soluble in water at allconcentrations below about 10% to 40%, depending upon the trans isomercontent.

More Detailed Description of the Prior Art

Today, the four types of iso acids used by the brewer are liquids,consisting of their potassium salts in water or propylene glycol. Solidsin the form of magnesium chelates have been substantially replaced bythe liquids in the last decade.

Because of differences in the concentrations at which the solutions of aparticular type of iso acid are most stable against precipitation, thefour acid types are sold in different concentrations in differentsolvent systems. IA is sold as a 30% solution of its potassium salt at apH of about 10 in water. DHIA is sold as a 35% solution of its potassiumsalt in water at a pH of about 10.5 and above, from which large,insoluble crystals of DHIA will precipitate over time. THIA is used as a5% or 10% solution of its potassium salt at a pH of about 9.5 to 10.5 inwater; and HHIA is not sold as an aqueous solution per se because of itslimited solubility. Because of the keto groups in their side chains,neither IA nor THIA form crystals from saturated solutions, but rathercan form gums at the bottom of the container upon cooling and standing.In these commercial preparations, the hop acids, and particularly 30% IAand 35% DHIA, as potassium salts at pH 10 or above in water, act asco-solvents for themselves. The co-solvent effect is demonstrated by theknown tendency to precipitate and separate at lower concentrations, asdiscussed below under the Westermann prior art. However, all forms ofhop acids can be solubilized in propylene glycol, as described in Todd(U.S. Pat. No. 3,486,906), and are available in this form, which alsoadds the advantage of increasing their dispersibility in soft water atpH 10 and above. Propylene glycol and ethanol solutions are the onlyforms of HHIA available, and their utility is impaired by therequirement of a solvent. The high trans products overcome the need touse propylene glycol or ethanol as a solvent. It should be noted thatsoft water must be used as the diluting agent for all potassium saltsolutions of the iso acids, since calcium and magnesium in the waterwill form chelates with hop acids and cause a haze and agglomerates andgummy precipitates. Below pH about 9 to 10 in deionized water, thedilute solutions of the prior art DHIA and HHIA do not form a clearsolution upon mixing but rather form gummy precipitates upon standing.The high trans products do not.

One common method of adding the hop acids post-fermentation is to dilutethem to a 1% or less concentration in soft water to which KOH has beenadded to bring the pH to 10 or above (Held, Master Brewers of theAmericas Association Tech. Quarterly, 35, 132-138, No. 3 (1998). Thehigh pH of the water is essential to prevent the formation ofprecipitates in the 1% dilute solution, and this has been ascribed toincomplete solubility of the hop acids in the dilute aqueous solution atlower pHs. These dilute alkaline solutions form hazes upon standing, andalso form precipitates causing haze after injection into beer or“stringers” of precipitates on the inside of a pasteurized beer bottle.The viscosities of the concentrated solutions make it impractical toinject them directly into beer, and in addition they tend to “shock out”and form particulate matter due to the rapid reduction of pH as they areintroduced, plugging the injection nozzle from time to time.

Solid magnesium chelates of IA are well described in Clarke, (U.S. Pat.No. 3,765,903 and 3,956,513). Others have added to his basic concept,but all IA chelates behave similarly. Chelates of DHIA and HHIA havenever been commercialized. The water-insoluble microparticulate solidchelates are added to water, in which they disperse as a cloudy hazewhich in turn is added to the unfinished beer. Other chelatepreparations are described in Humphrey (U.S. Pat. No. 3,875,316) andMitchell (U.S. Pat. No. 1,161,787).

Aqueous suspensions of solid micro particles of DHIA and HHIA aredescribed in Guzinski (PCT/US97/04070). These suspensions were made fromcommercial all cis products (p12, 1 23-24) made by the prior artprocedures described herein. They are suspensions. They had theadvantage over the prior art commercial solutions of DHIA in that theydid not require heating to about 80-90° C. to redissolve precipitatesbefore use. Indeed, one of the major advantages was the ability toredissolve the micro-particles by heating to about 60° C. Theredissolved solution was in turn diluted to 1% in soft water at a pH of10 prior to injection in the beer. Alternatively, the micro particlescould be added directly to pH 10 soft water preheated to about 50° C.,wherein they would dissolve and form a clear 1% solution within fiveminutes. The 1% solution, as in the case of the other prior artcommercial products, forms a haze upon standing (page 23, line 5), whilethe high trans product does not. Furthermore, his product, like otherprior art products, is not soluble at a 1% concentration in neutral softwater, whereas the products described herein are completely soluble. Andfurthermore, his product still required heating, albeit less vigorousthan 80-90° C. The novel high trans product can preferably be used atambient temperature, including brewhouse cellar temperatures of 10° C.or less. The commercialization of his product was abandoned because ofits limitations in practical brewing use, and particularly the need toheat it and the lack of clarity upon dilution.

DHIA is made from alpha acids by isomerization and reduction usingsodium borohydride, first described by Koch (U.S. Pat. No. 3,044,879). Asuperior process based on Koch was described in Westermann (U.S. Pat.No. 3,798,332), which used an extract made by his earlier invention(U.S. Pat. No. 3,558,326). Goldstein (U.S. Pat. No. 4,324,810) describesa method of making DHIA without the use of organic solvents. Today,manufacturers optionally separate the alpha acids from the remainder ofthe extract prior to isomerization and reduction, as described inGoldstein U.S. Pat. No. 4,767,640. These investigators produced theessentially all cis forms of the acids.

Todd (U.S. Pat. No. 4,002,683) describes an improved method forseparation of alpha acids and subsequent isomerization to IA, which isthe preferred method of separating alpha acids from an extract. A lessdesirable procedure for the separation of alpha acids from an extractand conversion to IA is given in Klingel, (U.S. Pat. No. 3,364,265), whoalso describes solid salts of IA. Mitchell (U.S. Pat. No. 3,949,092)describes a superior process. The method of separating and purifying thealpha acids is not critical to the disclosed process. The purity of thereduced product is a critical element. As the Examples also show, theratio of trans to cis isomers is very critical, and new to the art.

Procedures for making THIA from alpha acids are described in Stegink(U.S. Pat. No. 5,296,637), and Hay, (U.S. Pat. No. 5,013,571). THIA ismade from beta acids after the procedure of Worden, (U.S. Pat. No.3,923,897). HHIA is made by borohydride reduction after the method ofTodd (U.S. Pat. No. 4,666,731, Example 10), who employs less than halfthe molar equivalents of Worden, (U.S. Pat. No. 3,552,975) to achievereduction in a highly alkaline medium. Hay also describes the catalyticreduction of cis DHIA to make cis HHIA. HHIA, like DHIA, must besubstantially free of impurities if it is to form the novel product ofthe present invention. None of these investigators have suggested thisaspect of the present invention.

Guzinski (U.S. Pat. No. 5,200,227) describes mixtures of the prior artconcentrated aqueous products which, due to co-solvent effects, do notreadily crystallize. These had the advantage of physical stability overthe single acid products, but imposed limitations on the ratios ofdifferent acids which the brewer could add to a beer. Occasionally, itwas found that large crystals would form from these mixtures afterprolonged storage, but not to the extent formed in the single-acid formsof commerce. Because of the limitations on the ratios of the differenthop acids, they have limited utility. These products formed two phase,gummy solutions upon dilution in water, just as do prior art 35% DHIAsolutions. The novel forms of DHIA and HHIA described herein overcomethese limitations, since they are non-crystallizing and do not formgummy particulates.

Bavisotto (U.S. Pat. No. 3,615,660) describes the use of emulsifiers tostabilize DHIA extracts and make them suitable for adding to wort orbeer. The instant products overcome the need for the use of emulsifyingagents which end up in the beer, and the precipitation of the DHIAextract as the emulsion breaks upon addition to the beer.

Ting and Goldstein J. Am. Soc. Brew. Chem. 54, 103-109 (1996) describethe chemistry and purification of hop acids and their derivatives. Theirinvestigation examined specific pure cis and trans isomers. They furtherdescribed the physical properties of certain of these isomers. They didnot evaluate the solubilities of their pure compounds , including theircrystalline compounds and mixtures of them in water. They did not have,or suggest, the novel high trans isomer content aqueous solutions asdescribed herein, containing all of the analogues of the parent hop.

While the primary function of hops is to provide bittering to beer, asecondary function is to provide aroma. The aroma is derived from theessential oil contained in the hop cone. Aroma control is compatiblewith this invention by addition of hop essential oil to the kettle(preferably in the saponified extract described in Guzinski, U.S. Pat.No. 5,750,179). This invention also allows the addition of hop essentialoil to the DHIA and HHIA solutions, wherein it is sufficiently solubleto enable the brewer to add controlled amounts of essential oil to thefinished brew.

Held, cited above, summarizes the status of prior art hoppingmethodology.

OBJECTS OF THE INVENTION

The objects of this disclosure are to provide DHIA and HHIA having ahigh trans to cis isomer ratio and, as a consequence, to provide:

1. A non-precipitating solution of DHIA and/or HHIA.

2. Non-precipitating mixtures of DHIA and/or HHIA solutions with addedIA and THIA.

3. Hop acid solutions which do not form a haze or particulates upondirect injection into finished beer.

4. The analytical criteria which will provide quality assurance for theproducts, and which differentiates them from all prior art products.

5. The operational variables which may be adjusted by the manufacturerwhen making the novel products.

The Present Disclosure: A General Description of the Highly Soluble,High Trans Isomer Ratio Products of this Specification and ClearSolutions Thereof and a Discussion of the Most Relevant Prior Art

This specification discloses DHIA and HHIA having a high ratio of transto cis isomers and which form clear, non-precipitating aqueous solutionsof DHIA and HHIA, both of which are unknown to the prior art. This isdue to the heretofore unknown effect of the trans isomers in increasingthe solubility of the cis isomers. There is no explanation of thiseffect, which is contrary to the expectation that higher solute contentsdecrease solubility of all solutes. This effect is noticed in bothneutral and slightly (up to pH 10-11) alkaline water. Because of theimproved solubility in relatively low pH aqueous media and beer, theease of use and utilization in the brewery is vastly improved ascompared with the prior art cis products.

They do not form precipitates which must be heated to redissolve, orwhich must be filtered from the beer. They are soluble in soft water andtheir dilute solutions will not form hazes in the brewing cellarinjection tank. Because the purity of the hop acid must be high to makethem clear, they do not contribute an off-flavor “hang” to the beer, butrather possess only the desired fleeting bitterness withoutafter-bitter, especially on the palate. They can be directly injectedinto finished beer without forming haze or visible particulates,contrary to prior art products.

The preparation of the product critically differs from the prior art inthat the reduction is performed in an aqueous medium with sodiumborohydride (potassium borohydride is less preferred) at a pH belowabout 12, and preferably in the range of about 10 to 11, and attemperatures, times and concentrations which do not convert transisomers to cis isomers. Prior art products are made using a more highlyalkaline aqueous medium (pH 13.5), since it is well known thatborohydrides decompose readily if the water in which they are dissolvedis not highly alkaline. The presently-disclosed and critical procedureallows some borohydride to decompose due to the lower pH, while theremainder acts as a reducing agent. Buffers may be used to achieverelatively stable pHs during the reduction.

The effect of the lower pH on the DHIA or HHIA is to allow trans isomersto form without being changed to the cis isomer. It increases thecritical ratio of trans to cis isomers. Unless the trans isomer HPLCarea count is at least 10% of the cis isomer area count, and preferablygreater than about 20 to 30%, the product will not form a clear liquidaqueous solution at all concentrations from 1% to 20% and more. This iscritical to the invention. Prior art products have a ratio of trans tocis isomers of less than about 3% to 5% and, in most, trans isomers areundetectable. None of them will form clear solutions at concentrationranges of 10-20% in water, even at elevated pHs. The novel solubilityproperties of high trans isomer ratio containing DHIA and HHIA aredisclosed for the first time in this specification.

The preferred method also involves the reduction of IA rather than alphaacids. This increases the trans isomer ratio more than if a simultaneousisomerization/reduction is performed, as is the common prior artpractice. The simultaneous isomerization-reduction does not produce anacceptable product.

The most elegant prior art investigations of DHIA have been done at theMiller Brewing Co. laboratories.

The initial disclosure of a process for making DHIA is Koch, citedabove, filed in 1959. His examples use more than three to four times asmuch borohydride as the current art. Improved analytical techniques haveenabled his Miller successors to refine his basic process. Koch's DHIAproducts were dissolved in ethanol and added to boiling wort, so theyobviously were not suitable for post-fermentation addition.

The Westermann series built on Koch, and developed practical processesfor making DHIA using simultaneous isomerization/reduction. Moreimportantly, in U.S. Pat. No. 3,965,188, they showed how to make DHIAsolutions suitable for post-fermentation addition because of higherpurity than achieved by Koch, wherein “the purity is so high (at least90%) that the increase in turbidity is minimal”. (Col. 2, line 10 ff).His procedures, because of the use of SWS (12% NaBH4 in 40% NaOH), doesnot make a high trans DHIA but rather an all cis one, which will formprecipitates upon standing. This is why the high trans product cannot bemade by Westermann's U.S. Pat. No. 3,558,326. He claimed purities ofbetween 97.4 and 99.2%. Example 13 shows that his product is 77 to 78%rather than 99.2% DHIA by the standards described in this specification.Nor is it haze free, as is the product described herein.

It must be recognized that his “purities” were determined by the bestmethod available at the time, which consisted of extracting the “pure”DHIA from its alkaline solution into a water immiscible solvent,removing the solvent, and assaying the solids in alkaline methanol. Thestandard procedure at that time was to determine the absorbance at 254nm of an alkaline methanol solution of the solids, and calculate theDHIA content using some extinction coefficient (not mentioned in hisspecification). Regardless of the value of that coefficient, his solidswould have contained some humulinic acids, as well as other materialshaving absorbance at 254, and they would have been considered DHIA byhis assay. Furthermore, as shown in the comparative Example 11, hisproduct formed cloudy solutions at pH 10 in water, and curds andprecipitates at pH 7. It did not contain trans isomers.

Goldstein, following Westermann at the Miller Brewing laboratories, alsoperforms a simultaneous isomerization/reduction in U.S. Pat. No.4,324,810. He also uses SWS, a commercial 12% sodium borohydridesolution in 40% NaOH, and therefore his reduction is carried out underhighly alkaline conditions which cause only cis isomers to form. HisExamples 4 plus 5 show an overall yield of 82.7% of available DHIA witha purity of 96%. Not only was this an improvement on the yields ofWestermann, but he achieved his paramount objective of performing thereduction without the use of solvents other than water. Again, theprecise method by which he obtained his purity estimate of 96% is notgiven. As comparative Example 14 shows, his product was 74% DHIA vs hisclaimed 96%, by the state of the art techniques used in describing thepurity of DHIA in this specification. His product did not contain transisomers, nor did it form clear 1% solutions.

Goldstein in U.S. Pat. No. 4,767,640 separates the alpha acids from theextract, at a marginally higher pH than the critical pH of Todd, priorto isomerization/reduction without the use of solvents. He obtains animproved product, devoid of non-isohumulone light unstable products(NILUPS) found in the prior art products. (While Westermann claimedcomplete light stability, it is clear that the detection of instabilityhad progressed by the time of Goldstein's invention, and he was able toimprove the light stability of Westermann's products.) His product isclaimed suitable for post-fermentation addition to beer, but notspecifically for pre- or post- final filtration. This may be because hisproduct forms amorphous agglomerates and crystals on standing. It doesnot contain trans isomers. This is because his isomerization/reduction,as in Westermann, is conducted in a highly alkaline medium to startwith. Comparative Examples 15 and 16 describe his products. Injection ofhis products into finished beer cause insoluble precipitates to form.These are visible to the naked eye even after pasteurization.

While Goldstein prefers to avoid the use of solvents in his process,innocuous solvents such as hydrocarbons C-10 and below are useful inassisting the separations and purifications of the high trans products.They are not essential but rather optional and will assist in theremoval of the unwanted impurities, some of which are visible as posthop acid peaks in the HPLC. Others, such as “waxes”, may be undetectablein the HPLC assay. These must be substantially absent for the claimedDHIA and HHIA to remain clear in aqueous solutions when added to softwater.

Chicoye et al, in U.S. Pat. No. 4,759,941, describe a method for makingDHIA by treating hop pellets with borohydride. From his reactionmixture, he is able to separate an aqueous fraction which he adds postkettle. He makes no claim that it can be added to finished beer, andtherefore does not suggest the products described herein. Surprisingly,when pure alpha acids were reduced following his procedure, thereduction was incomplete and substantial impurities were formed. Perhapshis cellulosic materials act as a catalyst for the reaction to producehigh by-product levels in his procedure. Trans isomers were not detectedin his reactive product from alpha acids.

Guzinski's all cis microcrystalline product, which requires heating toredissolve, either by itself or in alkaline water, is clearly not arelevant prior art disclosure. Likewise, his slowly precipitatingmixtures of hop acids, which utilize their cosolvent effect, but are allcis isomers, do not suggest that the presence of trans isomers inhibitsand prevents the crystallization of cis isomers. Nor do the solids ofClarke. HHIA is not available as an aqueous product, since the all cisform, made by the Todd procedure (U.S. Pat. No. 4,666,731), is veryinsoluble.

Table 7-I in Example 7 summarizes the critical differences betweenproducts from the comparative Examples and the herein claimed process,as well as the effect a high trans isomer content has on solubility.Table 9-I in Example 9 shows the differences in performance of theproducts in beer.

SUMMARY OF THE INVENTION

What we believe to be our invention, then, inter alia, comprises thefollowing, singly or in combination:

A mixture of hexahydro-isoalpha acids (HHIA) or dihydroisoalpha acids(DHIA) having a ratio of trans to cis isomers greater than 10%.

And a mixture of hexahydro-isoalpha acids (HHIA) having a ratio of transto cis isomers greater than 10%;

such a mixture comprising hexahydro-isocoalpha acids,hexahydro-iso-n-alpha acids, and hexahydro-isoadalpha acids;

such a mixture wherein the ratio is greater than 20%;

such a mixture wherein the ratio is greater than 40%; and

such a mixture wherein the ratio is greater than 70%.

Also, such a mixture in the form of an aqueous solution of potassiumsalts of the HHIA, which solution forms a single phase liquid at a 20%concentration by weight of the potassium salts at a pH less than 9.5;

such a mixture wherein the solution forms a single phase liquid at a 10%concentration by weight of the potassium salts of the HHIA at a pH lessthan 8.5;

such a mixture in the form of an aqueous solution of the potassium saltsof the HHIA at a pH of 7 to 10.5 which is a single-phase solution whenat a concentration of 5% by weight;

such a mixture in the form of an aqueous solution of the potassium saltsof the HHIA at a pH of 7 to 9.5 which is a single-phase solution when ata concentration of 10% by weight; and

such a solution which, when diluted to a 1% concentration by weight indistilled water, forms a clear solution which does not form a haze uponstanding for six hours.

Also, such a mixture which contains less than 5% by weight of substanceswhich elute after the HHIA as detectable as area percent by HPLCprocedure;

such a mixture which contains less than 3% by weight of substances whichelute after the HHIA as detectable as area percent by HPLC procedure;

such a mixture which contains less than 1% by weight of substances whichelute after the HHIA as detectable as area percent by HPLC procedure;

such a mixture which contains less than 3% by weight of the HHIA ofsubstances which can be removed from an aqueous solution of the HHIA byextraction into a hydrocarbon solvent of 6 to 10 carbon atoms;

such a mixture which contains less than 2% by weight of the HHIA ofsubstances which can be removed from an aqueous solution of the HHIA byextraction into a hydrocarbon solvent of 6 to 10 carbon atoms;

such a mixture which contains less than 1% by weight of the HHIA ofsubstances which can be removed from an aqueous solution of the HHIA byextraction into a hydrocarbon solvent of 6 to 10 carbon atoms; whereinthe pH of the aqueous solution is below 10.5; wherein the pH of theaqueous solution is below 9.5; and wherein the pH of the aqueoussolution is below about 8.5.

Such a solution admixed with a solution of DHIA or with isoalpha acids(IA) or tetrahydroisoalpha acids (THIA);

such a solution containing glycerine, propylene glycol, alcohol, or hopessential oil; and

such a mixture in the form of solid potassium salts of the HHIAcomprising between about 10% and 70% trans isomers.

And a mixture of dihydro-isoalpha acids (DHIA) having a ratio of transto cis isomers greater than 10%;

such a mixture comprising dihydro-isocoalpha acids, dihydro-iso-n-alphaacids, and dihydro-isoadalpha acids;

such a mixture wherein the ratio is greater than 20%;

such a mixture wherein the ratio is greater than 30%.

Also, such a mixture in the form of an aqueous solution of potassiumsalts of the DHIA, which solution forms a single phase liquid at a 20%concentration by weight of the potassium salts at a pH less than 9.5;

such a mixture wherein the solution forms a single phase liquid at a 10%concentration by weight of the potassium salts of the DHIA at a pH lessthan 8.5;

such a mixture in the form of an aqueous solution of the potassium saltsof the DHIA at a pH of 7 to 10.5 which is a single-phase solution whenat a concentration of 5% by weight;

such a mixture in the form of an aqueous solution of the potassium saltsof the DHIA at a pH of 7 to 9.5 which is a single-phase solution when ata concentration of 10% by weight; and

such a solution which, when diluted to a 1% concentration by weight indistilled water, forms a clear solution which does not form a haze uponstanding for six hours;

Also, such a mixture which contains less than 5% by weight of substanceswhich elute after the DHIA as detectable as area percent by HPLCprocedure;

such a mixture which contains less than 3% by weight of substances whichelute after the DHIA as detectable as area percent by HPLC procedure;

such a mixture which contains less than 1% by weight of substances whichelute after the DHIA as detectable as area percent by HPLC procedure;

such a mixture which contains less than 3% by weight of the DHIA ofsubstances which can be removed from an aqueous solution of the DHIA byextraction into a hydrocarbon solvent of 6 to 10 carbon atoms;

such a mixture which contains less than 2% by weight of the DHIA ofsubstances which can be removed from an aqueous solution of the DHIA byextraction into a hydrocarbon solvent of 6 to 10 carbon atoms;

such a mixture which contains less than 1% by weight of the DHIA ofsubstances which can be removed from an aqueous solution of the DHIA byextraction into a hydrocarbon solvent of 6 to 10 carbon atoms;

wherein the pH of the aqueous solution is below 10.5;

wherein the pH of the aqueous solution is below 9.5;

wherein the pH of the aqueous solution is below about 8.5.

Such a solution containing glycerine, propylene glycol, alcohol, or hopessential oil;

such a mixture in the form of solid potassium salts of the DHIAcomprising between about 10% and 70% trans isomers.

Moreover, such a mixture of DHIA or HHIA which is in the form of asingle-phase aqueous solution of its potassium salts at a pH above about7.5 when at a concentration of 20% by weight.

Furthermore, the process of reducing (a) isoalpha acids (IA) to producedihydroisoalpha acids (DHIA) or (b) tetrahydroisoalpha acids (THIA) toproduce hexahydroisoalpha acids (HHIA), the DHIA or the HHIA producthaving a trans to cis isomer ratio greater than 10%, the reduction beingcarried out in an aqueous medium at a pH of about 8.5 to about 12.4using a borohydride;

such a process wherein IA are reduced to DHIA having a trans to cisisomer ratio greater than 10% using less than about 0.81 molarequivalents of a borohydride and a pH up to about 11.8;

such a process wherein THIA are reduced to HHIA having a trans to cisisomer ratio greater than 10% using less than about 0.81 molarequivalents of a borohydride;

such a process in which the temperature at which the reduction iscarried out is up to about 75° C. and in which the reaction isterminated before the trans to cis isomer ratio of the product DHIA orHHIA becomes less than 10%;

such a process wherein the reduction is carried out with up to about0.65 molar equivalents of borohydride;

such a process wherein the reduction is carried out with up to about0.55 molar equivalents of borohydride;

such a process in which a lower alkanol is also present;

such a process wherein the pH of the aqueous medium is buffered at about12.4 or below;

such a process wherein the buffering agent is selected from potassiumand sodium salts of phosphates, citrates, and borates;

such a process in which a non-reactive water-immiscible solvent is alsopresent;

such a process in which the water-immiscible solvent is a hydrocarboncontaining 10 or less carbon atoms;

such a process in which hydrocarbon-soluble haze-forming substances areremoved from the DHIA or HHIA product by admixing a hydrocarbon with theaqueous DHIA or HHIA phase and removing the hydrocarbon phase, whereinthe aqueous DHIA or HHIA phase is 15% or less DHIA or HHIA, and whereinthe pH is up to about 10.5, to give a DHIA or HHIA product wherein theremaining hydrocarbon-soluble substances are less than 3% by weight ofthe DHIA or HHIA product;

such a process in which the pH is about 7.5-9.5;

such a process wherein the hydrocarbon has 6 to 10 carbon atoms;

such a process in which the final aqueous DHIA or HHIA phase isconcentrated at a pH below about 10.5 and greater than 6 by evaporationof water, to give a concentrated aqueous phase containing between about5% and about 40% DHIA or HHIA;

such a process wherein the pH is between 6.5 and 8.5 and the DHIA orHHIA concentration is less than about 25%;

such a process wherein the borohydride is selected from the groupconsisting of sodium borohydride and potassium borohydride; and

such a process wherein the DHIA having a trans to cis isomer ratiogreater than 10% is subsequently converted to HHIA having a trans to cisisomer ratio greater than 10% by catalytic hydrogenation.

DEFINITIONS

Definitions Used in this Specification

As is known to the art, trans and cis isomers of the hop analoguesexist. Critical to this invention is the heretofore unknown effect of ahigh trans:cis isomer ratio on the solubility of the DHIA and HHIA. Inthis specification, this ratio is expressed as the % of the HPLC areacounts of the trans divided by the area counts of the cis isomers. Inprior art products it is well below 5% and usually almost zero.

One measurement of % impurities eluting after the hop acids in the HPLCprocedure is described below. It expresses the amount of haze formingsubstances, which are detectable by uv light, present relative to theamount of DHIA or HHIA. A second measurement relies on the extraction ofnon-absorbing haze forming substances with a water insoluble solvent, asdescribed in Example 11 below.

Procedures Used in Analysis are as Follows

Ultra-violet spectra (UV). For a whole extract, the American Society ofBrewing Chemists spectro procedure “Hops-6” was used. This entailsdiluting the test sample in alkaline methanol and running a scan, andusing a formula to calculate % alpha acids. This procedure is includedin the prior art references.

Absorption at 254 nm is a maximum for iso acids, and the strength of thesample is calculated on this basis using the extinction coefficient(E1%/1 cm). The sample is dissolved in alkaline methanol, the absorbanceat 254 nm determined, and the concentration calculated from theextinction coefficient. This procedure registers all absorbance at 254nm as the iso acid, and if absorbing impurities, such as humulinic acid,are present, it therefore overstates the true iso acid content. Only byHPLC can the true value of hop acid concentration be determined.

The extinction coefficient will vary for the various hop acids due todifferences in molecular weights, analogue composition, and thestandards historically used to determine them. For the purposes of thisspecification, the following numbers are used:

MW E1%/1 cm alpha acids IA 353 520 @ 254 nm DHIA 357 475 ″ THIA 361 480″ HHIA 363 460 ″ alpha 355 318 @ 325 nm

Experimental Method for HPLC Measurements

Hop extracts are diluted to a concentration of about 200-500 ppm totalhop acids in methanol. Separations are performed on a Waters 2690Separations Module with a 996 Photodiode Array. The HPLC column containsoctyl reverse phase packing (Zorbax Eclipse XDB-C8, 25×0.46 cm,5-micron) and was kept at 25° C. The aqueous buffer is 18:82 (v/v)acetonitrile:1% aqueous citric acid buffer (pH 7.0). The citric acidbuffer is prepared separately, adjusted to pH 7 with 45% KOH, andfiltered before combination with the acetonitrile. The mobile phaseprogram is given in Table D-1. Injection volume is 5 μL.

TABLE D-1 Mobile Phase Program for the HPLC Method Time (min) Flow(mL/min) % Methanol % Acetonitrile % Buffer  0 1 15 15 70  5 1 15 15 7030 1 80 15  5 33 1 80 15  5

The detector is set to measure the entire UV absorbance spectrum between230-400 nm with a resolution of 1.2 nm, filter response of 1, andsampling rate of 1 point/sec. HPLC plots are reported in “maxplot” mode,which reports the maximum absorbance value between 230-400 nm at eachpoint in the chromatogram. Data is analyzed by Millennium® 32 software(version 3.05.01, Waters and Associates). Maxplot chromatogram peaks arequantified with integration settings of threshold=15 μV/s, filterresponse=1, and minimum height and area=0.

The % impurities eluting after the hop acid is determined using the %area count at peak maximum. This is because many of the impurities donot have significant absorbance at 254, but peak in the range of 270 nmand above. An extinction coefficient is not needed for this calculation,as it only measures the total area under the peaks at the absorptionmaximum. The subject hop acids are identified in the traces, as well asthe peaks eluting after them, and the instrument calculates the areacounts. The relative area counts are independent of concentration of thesolution injected into the HPLC.

The cis and trans isomer peaks are defined in the HPLC traces of FIGS. 3to 6 for DHIA, and FIGS. 7 to 10 for HHIA. Since the prior art has notinvestigated the relationship of these peaks, the authors havedesignated these peaks as trans or cis, as defined in Example 10. Thedefinitions provided by the Figures show the critical differencesbetween the prior art and the novel products described in thisspecification.

Haze is measured by the American Society of Brewing Chemists procedureBeer 26.

Equivalents of a substance are molar.

Yields are based on an average molecular weight of the mixture ofanalogues.

Infra-red (IR) spectra are useful for demonstrating the differentchemical composition of the pure hop acid and the haze formingsubstances which do not absorb uv light. For the purposes of thisspecification, they are defined as “waxes.” These are isolated and thespectra described in Example 11.

GENERAL DESCRIPTION OF THE INVENTION

The Novel Process and Product

Examples 1 thru 4 show variations on the preferred process for making ahigh trans , highly soluble DHIA and HHIA which, in turn, do not formhazes upon dilution to 1% in distilled water..

It will be noted that none of these products form insoluble precipitateson standing, and that they may be added directly to soft water towhatever concentration the brewer desires. It will also be noted thatthey do not form precipitates visible to the unaided eye or measurablehaze upon direct injection into bottled beer. None of the prior artproducts have these qualities. Goldstein's NILUPs-free type DHIA all cisproducts and the all cis HHIA products presently available do not formclear solutions.

The purity of the hop acids must be exceptionally high if solutions ofthe high trans product are to remain clear. Substances eluting after thehop acid in the HPLC procedure must be less than 6%, preferably lessthan 4%, and most preferably less than 1% to 2%. Likewise, “waxes” whichdo not absorb uv light and which are hexane soluble, must be less than3%, preferably less than 2%, and most preferably less than 1%.

It is well known to the art that different hop varieties producedifferent ratios of the three major alpha acid analogues. The lowermolecular weight analogues have more solubility than the highermolecular weight ones. As a consequence, the upper concentration limitof the high trans products will vary with hop variety. Theconcentrations shown in the Examples are considered to be economical tothe brewer and suitable for any variety with which the authors arefamiliar.

The authors can offer no theory as to why a trans to cis isomer ratio ofabove about 10%, especially above about 20% to 30%, results in thegreatly increased solubility of the cis isomers, which, as mentioned inExample 6, are shown to be about 1.5% maximum for equally pure cis DHIAand 0.75% for cis HHIA. In some unknown manner, the trans isomersincrease the solubility of the cis isomers from about 1% in water to 10%and more. For example, a 20% solution of HHIA containing 4% transisomers and 16% cis isomers does not form crystalline precipitates. A16% solution of cis isomers does. As mentioned above, the solubility ofthe cis isomers alone is about 1%. The effect of the trans isomers uponthe solubility of the cis isomers' solubility is contrary toexpectation, since the higher the solute content, the lower should bethe solubility of related compounds. This effect cannot be a simpleresult of the analogue mixture, since the analogues are the same for thecis and trans forms. However, it is also preferred that the claimedproducts contain the approximate mixture of analogues found in theparent hop. Neither of these critical elements-the high trans isomerratio combined with all of the parent hop analogues—have grounding inthe prior art.

Edible anti-freeze substances, such as ethanol, propylene glycol, andglycerine may be added to the inventive products if they are to beexposed to below freezing temperatures.

In the process, buffering agents other than potassium phosphate may beused. These include sodium and other phosphates, as well as borates andcitrates. Details concerning the required process parameters arediscussed in the Examples.

When the claimed products are dried, they form amorphous solids whichcan readily be rehydrated to form aqueous solutions with the propertiesof the original aqueous solutions. Dehydration can be performed bytechniques known to the art, such as spray drying or by evaporation ofwater under vacuum or even at atmospheric pressure.

The claimed product is differentiated from prior art products by itshigh trans isomer content, the trans isomers being at least about 10% ofthe cis isomers (a trans to cis ratio of 10%), and preferably 20%, andmost preferably above 30%. It is further differentiated by the absenceof substances which elute after the DHIA or HHIA by HPLC analysis, suchsubstances consisting of artifacts and by-products of the reductionreaction. These substances interfere with the clarity of the aqueoussolutions of the products. The products are further differentiated fromthe prior art in that substances which are soluble in hydrocarbonsolvents and not detected by the HPLC procedure are essentially absent.

Furthermore, the products form stable single phase aqueous solutions atpHs substantially below the 10.5 to 11 minimums of the prior art, forexample between about 7 and 9.5, and are not dependent upon a 35% hopacid concentration, as shown by Westermann, to make a pourable liquidproduct. The solutions are stable at concentrations in the range ofabout 5% to 40%. While the instant products are preferably maintained ata pH below about 9.5, they are also stable at the pHs of the prior art.

In addition, the products form clear, non-hazing solutions in distilledwater at concentrations of 1% to about 5% and more. Prior art productsrequire raising the pH of the water to above 10 to effect dispersion ofa 1% solution, and even then haze forms upon standing. They form gumsand precipitates when added to distilled water. This simple test is onemeans of determining if the product meets the analytical requirementsdescribed above and in the claims.

The procedures by which these products are made combine elements of theprior art in a new way, so as to achieve the high trans ratio product.Unlike the Koch and Worden prior art, which uses about two or more molarequivalents of borohydride to achieve reduction and light stability, theherein disclosed novel procedure requires less than about 0.81 molarequivalents. Unlike the prior art conventional pHs of above 13 ofWestermann, Todd, Goldstein, and others, who also use less than 0.81equivalents, the pH during the reduction must be below about 12.4,preferably below about 12.2, and optimally below a pH of 11.2 or even10.6 for THIA. For IA, the upper pH should be below about 11.8, andpreferably below 11. While these pHs, which are well below the priorart, result in some borohydride decomposition which the prior art pHsabove about 13 to 13.5 deliberately avoided, the low pH is critical tothe trans isomer formation. The high pH of the prior art resulted inessentially all cis products, which are inherently of very lowsolubility. As little as 0.4 molar equivalents of borohydride may beused, but the range of about 0.55 to 0.65 is preferred. When the largeexcesses of borohydride, such as shown in Worden, are used, over-reducedand other by-products are formed and the actual yield of reduced DHIA orHHIA is so small as to make analysis problematic and the elimination ofhaze forming substances very difficult if not impossible.

Reaction temperatures below about 85° C. are feasible, the reactiontaking longer at lower temperatures. The preferred range is about 40° to75° C.

The reduction should be terminated before a significant amount of transisomer is converted to the cis form. This occurs more rapidly at highpHs and temperatures. The analytical procedures described herein providea guide to termination times.

Combining purification steps with the novel reduction conditionsdiscloses how heretofore unidentified haze and precipitate formingsubstances can be removed. These purification steps address substanceseluting after the DHIA or HHIA by the HPLC procedure (see Definitionsand Example 7 of this specification). These post-eluting substances musthave a total area count at peak maximums, according to the HPLCprocedure, of less than 5%, preferably less than 3%, and most preferablyless than 1% of the area counts of the hop acids.

In addition, there are also non-uv absorbing substances, undetectable bythe HPLC procedure, which must be critically less than about 3%,preferably less than 2%, and most preferably less than 1% of the weightof the hop acids.

Removal of these unwanted and unidentified substances, called “waxes” inthis specification, and not absorbing uv light, is preferably achievedby separating them from aqueous solutions at a pH below 10.5, andpreferably below about 8.5 to 9.5, and even as low as 7.5. Theconcentration of the hop acids in the aqueous phase during “wax” removalis less than about 20%, and preferably less than 15%. Because of theinsolubility of the all cis prior art forms at these pHs, separationswere done at elevated temperatures (Goldstein) or less than about halfof the DHIA was captured into the “clean” phase. As shown by thecomparative Westermann and Goldstein examples, yields were poor andsufficient impurities were present to cause haze and precipitation whendiluted in distilled water. It is speculated that the presence of thetrans forms assists in the separations, and therefore is critical to the“clean-up” procedure. The herein disclosed art gives yields in excess of75% and up to 85% to 90%.

Separation of unwanted substances is preferably effected using ahydrocarbon solvent, especially of C-6 to C-10, but other waterimmiscible solvents such as ether or methylene chloride may be used.Less preferably, they may be separated by allowing agglomerates of thesubstances to form, optionally in the presence of solid adsorbents suchas diatomaceous earth, and filtering the solids from the liquid phase.As with the water immiscible solvents, the solid separations areconducted at a pH below about 9.5.

DESCRIPTION OF THE DRAWINGS

Reference is now made to the drawings, wherein

FIG. 1 is a depiction of the structural formulas of cis and trans IA andDHIA.

FIG. 2 is a depiction of the structural formulas of cis and trans THIAand HHIA.

FIG. 3 is a trace of a HPL Chromatogram of typical prior art all-cisDHIA plotting absorbance units against time.

FIG. 4 is a trace of a HPL Chromatogram of trans DHIA plottingabsorbance units against time.

FIG. 5 is a trace of a HPL Chromatogram of high trans DHIA plottingabsorbance units against time.

FIG. 6 is an overlay of the chromatograms of FIGS. 4 and 5.

FIG. 7 is a trace of a HPL Chromatogram of a cis HHIA commercial productplotting absorbance units against time.

FIG. 8 is a trace of a HPL Chromatogram of trans HHIA plottingabsorbance units against time.

FIG. 9 is a trace of a HPL Chromatogram of high trans HHIA plottingabsorbance units against time.

FIG. 10 is an overlay of the chromatograms of FIGS. 7 and 9.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in both its method and product aspects, will bemore readily understood from the following detailed description,particularly when taken in conjunction with the drawings.

The following Examples are given by way of illustration only, and arenot to be construed as limiting.

EXAMPLE 1

Preparation of a liquid DHIA by reduction without the use of solvent inthe reducing medium.

52 g of a 29% aqueous solution of IA was added to 100 ml of water andthe pH adjusted to 10.5 to improve solubility. 0.87 g of NaBH₄ ( 0.55molar equivalents ) in 75 ml of water were added, the solution heated toabout 50° C., and the reaction terminated after two hours. 50 ml ofhexane, a C-6 hydrocarbon, was added, and the mixture cooled. Theaqueous and hydrocarbon phases were agitated with phosphoric acid at apH of 2.8, the aqueous phase containing boron discarded, and the organicphase was extracted with water to remove residual boron. The organicphase, containing the DHIA, was then partitioned against 100 ml ofwarmed distilled water brought to a pH of 8.4 with potassium hydroxide.(A pH of between about 7.6 to 8.8 is optimal for this separation,although higher and lower pHs may be used. Furthermore, concentrationsof 20%, preferably 15%, and most preferably about 10% or less in theaqueous phase are suitable for the partition). The aqueous phase wasreextracted with hexane to remove “waxes” and other insoluble material.The aqueous layer was then desolventized and concentrated to a 19%solution under vacuum. The 19% DHIA solution remained single phase,without precipitates, was 86% DHIA with 2.2% post-DHIA impurities asmeasured at peak maximums by HPLC. Approximately 11% of the area countat peak maxima was contributed by pre-DHIA peaks, which do not interferewith the solubility of the DHIA. The 19% aqueous solution was solublewith complete clarity at all concentrations in distilled water, and thetrans isomers HPLC area count was 33% of the cis isomer area count(trans:cis ratio equals 33%). The yield was 74%. Upon dilution to 10%with distilled water, the 19% solution remained clear, single phase, andwithout precipitates, unlike any prior art product.

The unexpected result of this procedure is the high percentage of transto cis isomers, well above that of about 0 to a maximum of 5% forcommercial DHIA and the prior art examples. This ratio is critical andis associated with the outstanding solubility at close to neutral pHs,as well as complete clarity in water and upon injection into beer (SeeExample 9). This product is new to the art.

A more difficult method of obtaining a desirably soluble DHIA, with ahigh trans isomer content, is to eliminate the use of a water immisciblesolvent during boron removal. This makes it more difficult to eliminateall the boron. Charcoal and silica gel and other adsorbents are usefulalternatives for water immiscible solvents for removing the substanceswhich detract from the clarity of the soluble DHIA solutions. Loweralkanols, such as methanol, ethanol, and isopropanol may be present inthe reaction medium. They will speed the reaction, but will alsodecrease the ratio of trans isomers.

Critical to the solubility and clarity of the DHIA is the highproportion of trans isomers, heretofore absent in DHIA products. Alsocritical to the clarity is the substantial absence of substances elutingafter DHIA by the HPLC methodology described in the specification, aswell as the absence of “waxes.”.

The prior art products, consisting of 35% DHIA aqueous liquids whereinthe hop acids are substantially free of trans isomers, as in the priorart examples below, are insoluble in water, and form a haze followingaddition to pH 10 water.

EXAMPLE 2

Preparation of DHIA from IA with the use of solvent.

500 g of a 25% (measured by total absorbance at 254 nm) aqueous solutionof crude IA was added to 250 ml of a 4% solution of NaBH₄ (0.75 molarequivalents). The pH was 11.4. 125 ml of limonene, a C-10 hydrocarbon,was added, and the mixture heated with agitation for 3 hours at 70-75°C. The mixture was cooled, and the organic phase separated from theaqueous DHIA phase. The organic phase was discarded. 200 ml of a lightpetroleum distillate (boiling point less than 100° C.) was added to theaqueous phase, and the solution acidified to pH 2 with phosphoric acid.The aqueous phase was separated and discarded. The organic phase waswashed once with water at a pH of about 3, and again at a pH of about 4to 5 to eliminate all boron. The organic phase was then partitioned with1000 ml of distilled water brought to a pH of 7.6 with potassiumhydroxide and the phases separated. The aqueous DHIA phase wasconcentrated under vacuum to 20% DHIA, during which process all residuallimonene was removed. The DHIA had a trans:cis ratio of 24% as measuredby HPLC area counts. The absorbance, as measured at peak maxima, of thepost-DHIA eluting substances was about 2.0 to 2.5% of the total DHIAarea count. (The post-DHIA eluting substances were about 9.9% in thecrude reaction mixture prior to partitioning. This shows theeffectiveness of the partition in removing the unwanted, haze formingimpurities). The yield was about 70% of the crude IA.

The 20% aqueous DHIA solution remained clear and without crystallizationfor more than three months. It formed clear solutions at allconcentrations when diluted in distilled water.

Lower alkanols, such as methanol, ethanol, and isopropanol may also bepresent, and they will accelerate the reduction reaction. Because oftheir cost, they are not preferred.

EXAMPLE 3

Preparation of a soluble HHIA.

280 g of a 42% pH 10 aqueous solution of commercial THIA ( 117 g=0.32moles) was added to 420 ml of distilled water. The resulting pH was 10.Then 7.4 g of potassium borohydride (0.195 mol=0.61 mole eq) was addedwith agitation, and the mixture was stirred for three hours while heatedto 70° C. The reaction mixture was cooled, 200 ml of hexane added, andthe pH reduced to about 2 by the addition of phosphoric acid withagitation. The lower acidic phase, containing boron, was discarded; thehexane phase washed once at a pH of about 2, and then with water.Although preferable, it is not essential that hexane be used during theboron removal procedure.

The HHIA was recovered from the hexane solution by partition into waterwith dilute KOH to a pH between about 6.5 and 8.5, preferably and inthis example about 7.5, to form an aqueous solution of HHIH. Theconcentration was about 10%. Its optional range is about 5% to 15% andless preferably 20% HHIA. The organic phase was separated from theaqueous HHIA phase and discarded. The aqueous phase and about 10% byvolume of hexane were heated to reflux with agitation. Temperature ispreferably elevated, and can be the reflux temperature of hexane orother water immiscible solvent, the reflux temperature being easilycontrolled. This assures that any residual insoluble substances areremoved from the aqueous HHIA phase. This includes “waxes”, which do notabsorb in the uv range and therefore are not detected by HPLC. (SeeExample 11). The organic phase was again separated and discarded.

The aqueous phase is then concentrated to any desired % HHIA by removalof water under vacuum, which also assures removal of any residualsolvent.

In this example, the resulting aqueous phase was 13.4% HHIA by uv. Thepeaks eluting after HHIA were 1.77% of the HHIA peaks by area count atpeak maximum, and HHIA was 92.1% of the area count at peak maximums. Thebalance was material eluting prior to HHIA, which does not interferewith the solubility. It had a trans:cis ratio of 98%. It did not formprecipitates on standing for three months.

The clear liquid 13.4% aqueous HHIA solution was made to 1% in distilledwater, pH 7.5, and was clear. The 1% solution did not form a haze orparticulates upon injection into beer at commercial use rates of 10 and20 ppm. Typical foam enhancement and flavor profile in the dosed beerswere observed by a trained panel.

HHIA with the above solubility characteristics and a trans:cis ratio ofabove 10% may also be made by catalytic hydrogenation of DHIA with atran:cis ratio of above 10%. However, this is not a preferred procedure.

EXAMPLE 4

Reduction of THIA to HHIA in a buffered system.

20 g of a 20% concentrate of commercial THIA, containing precipitates,and dark brown in appearance, was diluted with water to a concentrationof about 2% and the pH adjusted to 10. One-half volume of a 2.5%potassium phosphate solution containing two hydrogen equivalents ( 0.5molar equivalents) of NaBH₄ was added. The reduction was carried outover a three hour period, increasing the temperature from ambient to 70°C. during the course of the reaction. The resulting buffered pH wasbetween 11.7 and 11.9. Insoluble material formed during the procedure.At the end of the reaction, the aqueous phase was separated from theinsoluble gums, about one-half volume of hexane added, and the pHlowered to 2 with H₂ SO₄ with agitation. The aqueous phase wasdiscarded. The hexane phase was reextracted at pH about 3 and then withwater, pH 4, to remove residual boron. The hexane-organic phase was thenextracted into 100 ml of water by adjusting the pH to about 8 with 10%KOH. The aqueous phase was reextracted twice with hexane, which wasdiscarded. The aqueous phase was then rotovapped to provide a 22.5% HHIAsolution by uv. It assayed 85% HHIA by HPLC, with 0.44% post-HHIA peaks,14.6% pre-HHIA peaks, and the HHIA had a trans:cis ratio of 75%. Itremained as a clear, light tan solution without the formation ofcrystalline materials for more than three months.

The product was soluble in distilled water at all concentrations, anddid not form hazes upon standing. Prior art cis HHIA is soluble at <1%in distilled water. No concentrated aqueous solutions of HHIA areavailable to the present art.

While potassium phosphate is a preferred buffer, other buffers, such asmixtures of citrate and borate salts may be used. The advantage of usinga buffer is the maintenance of pH within a narrow range, thus avoidingthe effect of variations in pH as the unreduced form is converted to thereduced form, which has a different pKa.

EXAMPLE 5

Compatible single phase liquid, non-precipitating mixtures of solubleDHIA and HHIA with IA and THIA.

As has been noted, IA and THIA do not form crystalline precipitates,perhaps due to their molecular structure. Since the soluble forms ofDHIA and HHIA have been unknown to the art, this example is designed todemonstrate the limits of compatibilities of the mixtures. Guzinski U.S.Pat. No. 5,200,227 shows the limits of compatible mixtures of IA, DHIA,THIA, and HHIA which do not form precipitates of DHIA or HHIA onstanding. The objective of his invention was to overcome the tendency ofliquid solutions of DHIA and HHIA to form insoluble precipitates of thehop acids on standing. His formulations have found limited use in theart, since upon standing precipitates may form, particularly with cyclesof heating and cooling such as occur during transportation. Since hisDHIA and HHIA did not contain trans isomers, having been made by thethen commercial procedures, they were not the soluble non-precipitatingproducts of this invention.

Therefore it is necessary to determine the compatibility limits of thenew soluble products with IA and THIA, as is done in this example bycombining the different hop acids, as shown in Table 5-I.

TABLE 5-I Typical Mixtures of high-trans, clear soluble DHIA and HHIAand IA and THIA. Mixture Composition, % of Acid Total No. IA THIA HHIADHIA conc, % 1 30 70 18 2 50 50 20 3 50 50 20 4 60 40 20 & 35 5 57 29 1418 6 50 50 20 7 72 28 14 8 66 34 13

Mixtures of the DHIA and THIA and/or IA were compatible and remainedclear liquids at all ratios between about 1 and 99%. HHIA mixturesbehaved similarly. Since the high trans DHIA and HHIA are both clearsolutions, they may be mixed at any ratio and will remain clear.Preferred upper concentrations are below about 35% to 40%, and those ofgreatest ease of use in the brewery are between about 5% and 25%. Theconcentration of the mixtures is not critical to the invention, sinceany concentration adaptable to practice in a given brewery is feasible.

All solutions were clear upon dilution in water to 1%, except for themixtures containing 50% or more of IA and THIA. In the latter cases, thehaze was due to the IA and THIA, which formed hazes by themselves. Thesewere clear when added to pH 10 water. These mixtures do not form hazesor precipitates upon injection into beer.

In modern brewing, combinations of iso-acids are very useful indesigning foam, cling, and mouth feel characteristics into a beer. Theyprovide for much more flexibility than a single hop acid alone. Thesestable mixtures therefore offer a new and exceptionally practical mannerin which such mixtures can be utilized in the brewery.

EXAMPLE 6

Non-crystallizing limits of high trans water soluble DHIA and HHIA.

The essentially “wax” free products made by the procedure described inthe above examples were both concentrated and diluted to test theirsolubility ranges and to compare their performance with prior artproducts. The samples were allowed to stand at room temperature for afew weeks and, if crystallization occurred, it is noted below. The pHswere 7 to 8. Higher solubility limits are obtained at higher pH's thanthe range of 7 to 8 used in this example.

Concen- tration High trans DHIA High trans HHIA Prior art DHIA 30-40%Precipitates Precipitates 35% Precipitates Sometimes Prec. Slowly 20-30%Some precipitates No precipitates Separates and gums 15-20% Slightprecipitates No precipitates Separates and gums 10-15% No precipitatesNo precipitates Separates and gums 5-10% No precipitates No precipitatesSeparates and gums 1-5% No precipitates No precipitates Hazy and gums

No prior art product remains a single phase liquid upon standing atthese concentrations, at these low pHs, nor does it form a clearsolution when added to distilled water, as do the high trans products atthe noted concentrations. Prior art 35% DHIA, at a pH of 10 or above,acts as a cosolvent for itself, but will separate at lowerconcentrations and form gums and precipitates. Prior art HHIA appears tobe soluble at 5% or less in pH 10 to 11 water, although the solution isnot clear. Both the prior art DHIA and HHIA are insoluble in neutralwater and form hazes at 1% or less in distilled water at pH 10 andabove. Pure recrystallized cis DHIA has an upper solubility limit ofabout 1 to 1.5% in distilled water, and pure recrystallized cis HHIA'supper limit is about 0.75% even at pH 10. The presently inventiveproducts form clear solutions in distilled water at the concentrationsnoted above, as well as below 1%.

If a high trans DHIA or HHIA at a concentration above about 20 to 25%,for example 35% to 40%, is desired to minimize shipping costs, anyprecipitates can be dissolved easily by dilution and agitation.

It is obvious from the above that the high trans DHIA has very differentphysical properties than essentially all cis prior art DHIA. The priorart DHIA precipitates very slowly at a 35% concentration, and separatesinto two phases at less than about 30%. In complete contrast, the hightrans DHIA remains without precipitates at concentrations below about25%. The reason for this difference in physical and solubilitycharacteristics is unknown.

Since prior art all cis HHIA is only soluble to less than about 3% to 5%in water at pH above about 10, the enhanced solubility of high transHHIA as compared to high trans DHIA at concentrations of 20% to 30%cannot be explained. It is contrary to expectations, in that thehydrogenation of the acyl side chains should reduce its solubility ascompared to DHIA with less saturated acyl side chains.

In conclusion, the physical properties in the high trans products, ascompared to the prior art cis products, is not consistent with knownexplanations of solubility characteristics and is an unanticipated andcritical aspect of this invention. If the change in physical propertiesdid not occur, the high trans products would have no advantage over theprior art products. Likewise, that 5% or more cis DHIA or cis HHIA inthe presence of 1% or more trans DHIA or HHIA should be soluble andnon-precipitating is contrary to expectation, as the increased solutecontent should decrease the solubility of the cis forms. There is no apriori explanation of this behavior.

EXAMPLE 7

Summary of assay and analytical data of clear, highly water soluble,non-precipitating products and prior art products.

It was mentioned in Westermann -188 (col 3, lines 66 to col 4 line 5)that 5% solutions of his DHIA form an aqueous and oil phase on standing.He found, to his surprise, that when concentrated to the range of aboveabout 35% they remained as a stable single phase solution. Thisco-solvent effect of the hop acids for themselves at relatively highconcentrations is also noted in Guzinski -227, who points out thatprecipitation occurs over time with hard to dissolve crystals.

The new high trans ratio highly water soluble forms of DHIA and HHIA donot depend upon their cosolvent effects for their liquidity, but ratherform clear solutions at lower and higher concentrations. They are notdependent upon a cosolvent effect created by a 30% or 35% organiccontent in the solution. Typical performance at various concentrationswas evaluated and the results are shown below. This demonstrates theabsence of haze formation when diluted in water. The Table also offerscomparatives with prior art Examples in this specification, as well aswith a commercial DHIA. As noted in the prior art section, HHIA of theprior art cannot be provided in a usefully high concentration in water,but is provided as a propylene glycol solution.

The suspension evaluated was a microparticulate of Guzinski(PCT/US97/04070).

TABLE 7-I Comparative Properties of high trans “soluble”, suspensions,and liquid hop acid products. Example % 1% in No. Type trans:cisImpurities water  1 Soluble 33 0.2 Clear  2 Soluble 24 <2.5 Clear  3Soluble 98 1.8 Clear  4 Soluble 75 0.5 Clear  5 Soluble >10 — ClearMixtures 12 Prior Art <1 6.5-13 Milky, Curds 13 Prior Art 0 15 Milky 14Prior Art 0 7.1 Milky 15 Prior art 0 >5 Milky Micro— 0 5.7 Hazyparticulate Commercial 0 >5 Milky 35%

Conclusions:

Only the products with a trans to cis ratio of substantially greaterthan the prior art form clear solutions in distilled water, and remainhaze free. While the absolute lower limit of the trans to cis ratio hasnot been established because not all analogue combinations have beenevaluated, if it is above about 10-20% the non-crystallizing clear watersoluble characteristic is achieved. Ratios of about 10% work incombination with IA and THIA. There is no upper limit, and ratios aboveabout 30-40% and especially 50% are preferred, especially for HHIA.

While not affecting the solubility of the DHIA and HHIA, the impuritieshave an important effect upon the clarity of the 1% solution.

Therefore the optimal product has a trans ratio above 10%, andpreferably above 20%, and especially above 30%, The DHIA and HHIAimpurities eluting after the hop acid by HPLC, as measured by area countat peak maximums, are desirably less than about 8%, and especially lessthan about 5%, and most preferably less than about 3%, and mostdesirably less than 1%.

EXAMPLE 8

Flavor comparison with prior art products.

The bitterness profiles of the high trans products were shown to be thesame as those of the existing commercial types by two techniques, bothof which involve dilution of the test material in water at aconcentration of 15 ppm.

The first is a triangle test, in which individuals are asked to selectthe odd sample among three samples presented to them. The results arethen subjected to statistical analysis to determine if the samples aredifferent within a 95% confidence limit. This test showed that there wasno difference in bitterness between the commercial products and theinventive samples.

The second test is more sophisticated, and tells whether or not the hopacid has the same fading characteristics in the mouth. It is called atime-intensity analysis, and involves the subject tasting a sample inwater, and recording the bitterness impression at five second intervals.This provides an analysis of the maximum bitterness perceived, as wellas the manner in which the bitterness disappears in the mouth. Theinventive high trans to cis ratio sample had the same perceivedintensity as one commercial DHIA sample, which was more bitter than asecond commercial DHIA sample, but otherwise had the same type of fadingcurves. All had similarly shaped time intensity curves. High trans ratioHHIA samples also had the same time intensity curves as all cis HHIA.

There is no a priori reason that a high trans ratio product should havethe same flavor characteristics as present commercial products, since itcontains a critically higher ratio of trans isomers than do existingcommercial products, which are all cis isomers. It is well known thatdifferent isomers of a substance have different smells and tastes, andthe result shown in this example is not predictable by theory.

It was noted that both the DHIA and HHIA commercial samples had anastringency not associated with pure cis DHIA and HHIA in the timeintensity analysis. Commercial DHIA has been noted to contributeastringency in light beers, where it is more obvious than in regularbeers containing more malt derivatives.

EXAMPLE 9

Comparative clarity and solubilities upon injection into beer.

The products of the above examples were directly injected into acommercial beer at brewing cellar temperature of about 4-8° C . Becausethe Guzinski PCT suspensions were too thick to inject directly, allformulations were diluted to 1% concentration prior to injection. Theprior art products, including the microparticles of Guzinski, did notform clear solutions upon standing, even though the pH of the solutionswas above 10, and was substantially above that of the high trans to cisisomer ratio DHIA and HHIA solutions (pH about 7 to 8). Followinginjection of 20 ppm of the hop acid, the bottles were shaken, heldovernight, and haze measured using a clear glass bottle. The increase inFTU haze units is shown below in Table 9-1.

TABLE 9-1 Behavior of products upon injection into beer. Aq. In BeerSolution Example Product FTU Precipitates Clarity HHIA Micro 20 Yes HazyParticulate  4 Trans:cis 0 No Clear 32% DHIA Commercial 50 Yes Hazy 12Westermann >50 Yes Cloudy 15 Goldstein 30 Yes Very hazy  1 Trans:cis 0No Clear 20%

Mixtures of the high trans ratio DHIA and HHIA solution with IA and THIAdo not affect the turbidity of the resulting beer unless the IA and THIAare impure and create turbidity by themselves. In that case, theincrease in FTU is caused by the IA and THIA.

Conclusions: The prior art products produce significant haze when dosedinto beer. This behavior is exhibited even when they are dosed in as 1%solutions at pH 10, whereas the high trans solutions of the hereindescribed products are not affected by the pH of the dosing solution.This makes the high trans:cis ratio DHIA and HHIA preparations uniquelysuitable for post-final filtration addition to beer. As a consequence,the hop acids are not removed along with the haze forming substancesupon filtration, and the utilization (recovery in the beer) will be inthe 90-100% range rather than 50-70% range.

EXAMPLE 10

Comparison of HPLC differences between commercial all cis hop productsand the novel high trans isomer DHIA and HHIA products.

FIG. 3 is an HPLC trace of a typical prior art all cis- DHIA. FIG. 4 isa trace of trans DHIA, which contains a very small amount of cis. FIG. 5is a trace of the new, highly soluble, high trans DHIA. FIG. 6 is anoverlay of FIG. 4 and FIG. 5, in which the solid line is the transproduct, and the dotted line is the new high trans soluble product.

FIGS. 7, 8, 9 and 10 show the comparable traces for HHIA. FIG. 7 is thetrace of the prior art HHIA, FIG. 8 of all trans HHIA, FIG. 9 of the newhigh trans HHIA, and FIG. 10 an overlay of FIG. 7 and FIG. 9.

The peaks designated as trans in FIG. 4 and FIG. 8 were made byseparating trans IA and THIA by procedures known to the art, andreducing the trans IA and THIA under the conditions of Examples 1 to 4,which do not epimerize trans isomers to cis isomers. These reducedproducts contained essentially only the trans peaks. It is known thattrans isomers convert to cis isomers upon refluxing at pH 12.5 in water.Their identity was further confirmed by using this procedure to producethe cis forms.

It is obvious that the new high trans DHIA product of FIG. 5 has a largepeak, eluting at about 12½ minutes, which is present as a shoulder inthe all cis product of FIG. 3, and essentially absent in the all transDHIA of FIG. 4. This peak is an n-analogue cis-stereoisomer whichbecomes the 16 minute n-analogue cis isomer upon heating at a pH abovethat used to make the high-trans product. It will be noted that thispeak is very evident in the overlay of FIG. 6. Its presence does notaffect the identification of the trans peaks. Likewise, the 9 minuteco-cis isomer is more slowly transformed to its 12 minute co-cis isomerat an appropriately higher pH.

The differences in the new products and prior art products are clearupon examination of the overlays.

It is the area count of the trans peaks, divided by the area count ofthe cis peaks, multiplied by 100, which gives the per cent trans:cisratio.

The above chromatograms were run on the same instrument, with the sameeluting solvent and column, on the same day. It is well known that theretention times of the individual compounds will change with changes incolumn conditions. However, the relative positions of the peaks will notchange if the same type of column and eluting solvent is used. (See“Experimental Method for HPLC Measurements”.)

Peaks appearing after the last trans isomer are those which areconsidered post- DHIA and HHIA impurities. Since there are many of them,all with very low area counts in these purified products, they look andare insignificant. In unpurified product, such as commercial DHIA, or anunpurified product, they would be clearly visible and represent about 8%to 15% and more of the area count.

EXAMPLE 11

Separation and analysis of “waxes”, and haze contributed thereby.

One group of substances which cause a haze when the inventive product isadded to water at a pH below about 8 does not absorb in the uv orvisible spectra, so they do not show up in an HPLC purity analysis. Forthe purposes of this specification, they will be called “waxes,”although their chemical composition remains unknown. Separation, hazeformation, and infra red spectral analysis of the “waxes” is describedbelow.

A. Separation of Haze Forming Substances from HHIA

227 g of a 20.7% clear solution of HHIA (44.6 g of HHIA) with a %trans:cis ratio of 77, and which formed a hazy solution when added towater at a 1% concentration was the starting material. It was agitatedin sufficient water to give a 10% concentration, which was also hazy.The pH was adjusted to 8.3 with KOH and the temperature raised to 80° C.with agitation to effect clarification, cooled to about 60°, andagitation continued while 112 ml heptane was added. It was stirred for10 minutes. The aqueous phase was separated from the interphase andheptane phases, and the combined interphase precipitates and heptaneevaporated to dryness to give 7.72 g. This contained 3.3% HHIA by uv.This in turn was dissolved in methylene chloride, which was back washedthree times with alkaline water to remove the trace of HHIA, and analiquot of the methylene chloride solution evaporated to dryness. These“waxes” were submitted for IR analysis.

The aqueous phase was concentrated to 12% HHIA, which removed residualheptane. It formed a clear 1% solution in distilled water, without theformation of hazes upon standing. This demonstrates that the hazeforming “waxes” had been removed, and the HHIA will not form haze uponinjection into beer as a 1% aqueous solution, which indeed is the case.The “waxes” by themselves caused a haze measured at 30 FTU when 2 ppmwere injected as a 1% alcoholic solution into beer.

Discussion:

The conventional dewaxing is done by separating insoluble materials fromsolutions of HHIA at concentrations above about 15% to 20%. To removethe last traces of haze forming substances, it is preferable to dilutethe HHIA to less than 15%, preferably 12-14%, and most preferably belowabout 10% concentration. Unless the solutions are dilute, more thanthree extractions of the aqueous phase with the organic solvent arerequired.

It is considered, based on this experiment, that the HHIA acts as acosolvent for the “waxes,” which is why HHIA concentrations of less thanabout 15% facilitate their removal This is why they have not beensuccessfully removed from the prior art all cis isomer commercialproducts.

B. Separation of a Mixture of DHIA and Haze Forming Substances

A pH 7.8 DHIA solution with a % trans:cis ratio of 42 was extractedtwice with hexane at a concentration of about 15% and the aqueous phaseseparated from the organic phases. The organics were discarded and theaqueous phase concentrated and desolventized to give a clear 20.7%solution. 227.6 g of this clear solution was diluted to a 10%concentration in water. The solution remained clear. Upon dilution inwater to 1%, it developed a very slight haze. It was then warmed to 60°C. to effect dissolution of any residual “waxes” which had beendissolved in the clear 20.7% solution due to the high DHIAconcentration, 100 ml of hexane was added, the mixture stirred tenminutes, and then cooled. The interphase and hexane phases wereevaporated to dryness under vacuum, and 0.42 g of solids recovered.These assayed less than 0.89% DHIA by uv. The solids were dissolved inmethylene chloride and backwashed three times with alkaline water toremove any residual DHIA. The solution was evaporated to dryness and thesolids submitted for IR analysis.

The separated aqueous phase was reduced in volume under vacuum to removeresidual hexane, and diluted to 10% and 1% in distilled water. No hazeformed upon standing, showing that the haze forming waxes had beenremoved. Injection of the 1% DHIA solution into beer did not cause anincrease in FTU at 10 ppm, whereas the waxes caused an increase ofbetween 10 and 20 FTU at 2 ppm when injected as a 1% alcoholic solution.This again demonstrates that when the purified product does not throw ahaze in dilute aqueous solutions, it will not cause a measurable hazewhen injected into beer. After the aqueous solution was concentrated to12% DHIA, it remained as a clear solution and did not form precipitates.It produced a clear 1% solution, without later haze formation, indistilled water, thereby demonstrating that the haze-forming substanceshad been removed. Upon injection of 20 ppm as a 1% alcoholic solutioninto beer, no haze was formed as measured in FTU, and none was visibleto the eye.

Discussion.

Concentrations of 25% and more have been used in conventional processesfor removing impurities from these hop acids. This solubilization ofwaxes has not been noticed by the prior art, and explains why prior artworkers have attributed residual haze as due to the solubility limits ofcis DHIA and HHIA, which indeed are in the 1% range for their all-cisisomer forms. This experiment shows that the solubility of “waxes” inthe more concentrated high trans:cis isomer ratio products causes hazeformation unless they have been removed. Optimal pH ranges for waxremoval are between 7.5 and 10.5, the range 8.5 to 9.5 being best.

It is also apparent that the removal of the “waxes” is required if clearHHIA and DHIA solutions are to be formed. If more than about 2% to 6% ofthe haze forming waxy substances are present, both HHIA and DHIA willform hazes and their concentrated solutions will form gummy sediments onstanding. Therefore to prevent separation, a “wax” content, as a percent of the hop acid, of less than about 6% is an essential. To form ahaze free solution, less than about 2%, and preferably less than about1%, is a requirement. The level at which they may be tolerated is, ofcourse, determined by the increase in haze which is acceptable to thebrewer.

While the use of a water immiscible solvent is a preferable means of“wax” removal, if a brewer can tolerate some increase in haze it isfeasible to eliminate the solvent and either filter off the insolublematerials from a dilute hop acid solution, or allow them to form asediment. This may result in a wax content of up to about 6%, which willcause an increase in haze in the finished beer. However, if the beer isalready hazy, this may not be harmful.

C. HPLC Analysis and Infra Red Spectral Analysis of the “Waxes”

HPLC separations and IR spectra were run on both the DHIA and HHIA“waxes”.

The “waxes” did not contain measurable amounts of DHIA or HHIA by HPLC.

The IR spectra were obtained on a Perkin Elmer 1710 FTIR spectrometerusing thin films of the “waxes” cast on KBr windows. The spectra showedthe presence of OH groups at 3200-3600 and 1000-1300 cm⁻¹; aliphatic CHgroups at 2800-3000 cm⁻¹, and carbonyl CO groups at 1600-1750 cm⁻¹. TheOH and carbonyl CO stretching bands are greatly enhanced in pure samplesof DHIA and HHIA, while the “waxes” have significantly more absorbancebetween 2800 and 3000 cm⁻¹. This is interpreted to mean than the “waxes”contain substantial amounts of aliphatic hydrocarbon moieties in theform of fatty alcohols, which are absent in the pure hop acids. Thesealiphatic hydrocarbon moieties explain the haze forming potential of the“waxes”.

EXAMPLE 12

Comparative with Westermann U.S. Pat. No. 3,558,326 and 3,965,188

Westermann builds on the work of Koch, who showed DHIA in alcoholicsolutions which, when added to wort, improved the light stability ofbeer. Westermann's objective was to provide a DHIA fraction suitable forpost-fermentation addition, and of improved purity.

His first patent showed an improved process for making DHIA, and hissecond patent took that a step further and separated fractions ofincreasing DHIA purity for post-fermentation use.

This example shows why Westermann does not anticipate or suggest thisinvention, since his product had neither the purity he claimed, wouldnot perform like the product of this invention, nor had the samephysical properties.

In his procedures, he prepares a feed stock in −326 which is used to hisExamples 6 to 16 of −188 to demonstrate optimum pH and concentrationranges for separation of his “pure” DHIA from his feed stock.

The procedure of −326 was followed on a bench scale by combining 50 g ofa supercritical CO2 hop extract (46.5% alpha by uv, 23.25 g), 175 ml ofwater, 75 ml of hexane, and 13 g of commercial SWS (12% NaBH₄ in 40%NaOH, 0.63 molar equivalents). The pH was 13.5. The mixture was heatedat 60° C. (140° F.) for three hours, cooled, and acidified to pH 2 withdilute H₂SO₄. It was agitated warm for one hour, and the phasesseparated. The hexane-DHIA phase was washed again with acidic water, andthen with water to remove residual boron and acid.

The hexane-DHIA phase was made to 140 ml with hexane, and 20 ml aliquotswere withdrawn for the −188 experiments 11-15, which provided his bestyields and most “pure” product. Each aliquot is calculated to containabout 3.32 g of DHIA.

To the hexane solution was added 20 ml of distilled water, and the pHadjusted upward with a small amount of dilute KOH to the target pH. Theaqueous phase was separated from the hexane phase, which containedresins, beta acids, and other unwanted substances.

In order to determine the purity of the DHIA in the cloudy aqueous phase(Westermann does not report his assay technique, but in any event modernmethods were not available), the aqueous phase was extracted withmethylene chloride (10 ml) at a pH of 2-3. At this pH the hop substancesare extracted into the solvent. The methylene chloride was separated,removed under vacuum, the solids weighed and then assayed by uv andHPLC.

The results are reported in Table 12-I.

TABLE 12-I Purity of Westermann's DHIA according to present methods, ascompared with Westermann's reported purities. Westermann's % % % ClaimedThis experiment His by by post % % Yield by No. pH wt,g uv HPLC impurPurity Conc. uv HPLC 11 5.9 0.888 90.5 79.4 7.4 95.5 33.4 26.7 21.2 126.5 1.126 95.2 78.3 6.5 97.4 47.4 33.9 26.7 13 7.0 1.240 92.2 76.9 7.399.2 51.8 37.5 28.7 14 7.5 1.246 80.3 77.9 12.4 90.7 59.2 28.7 26.8 157.95 1.388 79.8 77.1 13.4 85.1 69.2 33.4 32.2

As will be noticed, the weight amounts extracted into the water increasewith the pH, as did those of Westermann. This is because the solubilityof the hop acids in water increases with pH. The uv purities are belowhis, being measured by the total absorbance at 254 nm. The HPLCpurities, as is expected, are below the uv purities. This is because theHPLC separates the impurities, such as humulinic acid and alpha and betaacids, from the DHIA. Only the DHIA content is used in the HPLC puritycalculation. Since humulinic acid has a lower molecular weight thanDHIA, and is extracted into water at a lower pH, a higher concentrationof that acid will result in a higher uv “purity”, such as claimed byhim. It is known that the retention times of the individual compoundswill change with changes in column conditions. However, the relativepositions of the peaks will not change if the same type of column andeluting solvent is used.

Peaks appearing after the last trans isomer are those which areconsidered post- DHIA and HHIA impurities. Since there are many of them,all with very low area counts in these purified products, they look andare insignificant. In unpurified product, such as commercial DHIA, or aslightly impure product, they would be clearly visible and representabout 8% to 15% and more of the area count.

The peaks designated as trans in FIGS. 2 and 5 were made by separatingtrans IA and THIA by procedures known to the art, and reducing the transIA and THIA under the conditions of Examples 1-4, which do not epimerizetrans isomers to cis isomers. These reduced products containedessentially only the trans peaks. It is known that trans isomers convertto cis isomers upon refluxing at pH 12.5 in water. Their identity wasfurther confirmed by using this procedure.

If his “purity” was determined by total absorbance at 254, thedifference can be accounted for by using a higher extinction coefficientthan that used in this specification. The trans to cis isomer ratio wasless than about 0.1%, so his product is essentially cis.

A high per-cent of post DHIA impurities, as measured by HPLC at peakmaximum, along with “waxes”, will cause the formation of gummyprecipitates in a liquid DHIA solution, even if the product has a hightrans:cis ratio, which his product does not.

All of the above products form gummy precipitates in water at pH 7 andform a very cloudy solution at a 1% DHIA concentration at pH 10-11.

EXAMPLE 13

Comparative with Goldstein -810 Examples 4 plus 5.

50.7 g of CO2 extract at 46.5% alpha acids by uv (23.6 g) were combinedwith a solution consisting of 500 ml water, and sufficient SWS toprovide 2.2 mol of NaOH and 0.75 molar equivalents of NaBH₄ per mole ofhop acids. The pH was 13.5. The mixture was agitated three hours at60-65° C. It was cooled slightly, and sufficient 50% H₂SO₄ added withagitation to drop the pH to about 2. The acidic phase, containing boron,was discarded, and the oil phase washed again at a pH of about 2, thephases separated, and the oil phase washed again with water. Theseparated organic layer weighed 35 g and assayed 62.8% DHIA by uv.

To 27.5 g of the organic layer was added 31 ml of 1N KOH, which broughtthe pH to 7. The mixture was agitated at 60-65° C. for 30 minutes. Theaqueous DHIA phase was separated from the oil phase. The aqueous phaseassayed 46.2% DHIA by uv, and the solids extracted therefrom were 74.1%DHIA by HPLC. The impurities eluting after DHIA were 15% of the areacount at peak maximum, far above the critical limit of this invention.Goldstein reported 96% purity for the solids in the aqueous phase, butdid not report his assay procedure. The explanation provided for asimilar purity discrepancy in Westermann is applicable here. No transisomers were present, and his product formed a very cloudy solution at1% in pH 10-11 distilled water. The product itself formed gummy crystalsand solids upon standing. It is not suggestive of the presentlyinventive product for the same reasons as the product of Westermann.

EXAMPLE 14

Comparative with Goldstein-640.

This example follows the procedure of Goldstein-640. Examples 2 and 3,except that it was performed on a bench scale. The amounts of reagentsused and the procedures were:

300 g of hop extract, 46.5% alpha acids by uv, and 45% KOH (46.6 g) wereagitated and warmed to about 45 deg for about 5 minutes, placed in aseparatory funnel, and the lower somewhat alkaline aqueous phasecontaining the alpha acids was separated. It contained 19.4% alpha byuv, as compared to Goldstein's 16.8%.

46.6 g of the aqueous alpha acid phase were added to 0.5 mol equivalentsof NaBH₄ (0.48 g) in 7.1 ml of water containing 1 mol equivalent of NaOH(1 g). The pH was 12.7. The reaction mixture was agitated at 175° F.(79° C.) for 2 hours.

The boron was removed from the aqueous phase by acidification with 50%H₂SO₄ with agitation, separation of the lower aqueous layer from theupper acidic DHIA oily phase, and by rewashing of the oily phase at a pHof about 2 with water, and then with water alone. The separation andagitation was facilitated by heating to reduce the viscosity of the oil.

6.7 g of the acidic DHIA was agitated with water and the pH adjustedwith dilute KOH to make a 40% DHIA solution by uv at pH 7.0. It wasagitated at 50-65° C. for one hour. The aqueous phase was allowed toseparate from the NILUPS oil phase as the mixture cooled, and the phasesseparated in a funnel. The DHIA phase assayed 37% DHIA by uv. The HPLCshowed that this consisted of 83.4% DHIA, and 7.1% post-DHIA peaks byarea count at peak maxima. It was not “pure” DHIA. It did not containtrans DHIA.

The warm liquid DHIA phase separated into two phases, with amorphoussemi-crystalline solids appearing overnight. It was reheated to dissolvethe crystals prior to evaluation in water, in which it formed a hazydispersion at pH 10 and concentration of 1%. It formed a two phasesolution on cooling before crystals appeared. It formed a hazydispersion in distilled water.

It will be noted that there are major discrepancies between the uv andHPLC assays of DHIA in this example, as in Westermann. This is becausethe uv procedure measures concentration at a single wave-length, 254 nm,at which impurities absorb uv light. Therefore the more impure theproduct is, the greater the discrepancy will be. A product assaying 98%by uv can be much, much lower by HPLC due to impurities absorbing at 254nm and counted as DHIA in the uv assay, and separated out and notcounted by HPLC.

EXAMPLE 15

Comparative with Goldstein -640 Example 1 plus 3.

The procedure in his Example 1 was followed, which differs from hisExample 2 in that about 0.74 instead of 0.5 mole equivalents of NaBH₄ isused for reduction.

The acidic DHIA-NILUP oil phase was separated from residual boron usingacidic water. It was titrated with dilute KOH to a pH of 7.0-7.2., as inhis example. The aqueous DHIA phase was separated, after coolingovernight, from the NILUP oil phase. The DHIA aqueous phase assayed41.5% DHIA by uv, and the DHIA was 80.2% of the HPLC 254 nm area count.The acid form as recovered in his Example 3 assayed 78.2% DHIA, and 8.2%post-DHIA by area count at peak maxima. He does not provided HPLC assaysfor these final products. Trans DHIA was not detected. Yields wereconsistent with those reported by Goldstein.

The warm liquid DHIA phase formed amorphous solids overnight. It wasreheated to dissolve the crystals prior to evaluation in beer in Example9.

EXAMPLE 16

Discussion of effect of varying conditions upon the reduction .

The conditions which produce a high trans product are similar to thoseof the prior art, in that reaction times, temperatures, and pHs, andequivalents of borohydride are found. The reason that the high transproduct has not been produced in the prior art is simply that the rightcombination of conditions has not been used. Indeed, the thrust of theart has been to produce the 35% all cis DHIA first described byWestermann. The prior art conditions under which the reductions wereperformed were not critical, in that the principal objective was to makesure that essentially all IA was reduced and the light stabilityimproved. This meant that high pHs and temperatures, as well as longreaction times, gave acceptable yields without residual IA, since cisDHIA is stable at high pHs. Furthermore, as over-reduced and otherby-products and degradation products do not have the reactive keto groupin the molecule, they are light stable. And since humulinic acid typeand other degradation products were measured as DHIA by uv analysis,yields as measured by uv at 254 nm were considered acceptable.Commercial production of HHIA essentially follows the procedure of Todd(U.S. Pat. No. 4,666,731), which is similar to Westermann, in which SWS(12% sodium borohydride in 40% NaOH) is used, and the pH during thereduction is above 13. The products are all cis isomers.

The high pH reduction will not make either trans DHIA or trans HHIA.This is because they “epimerize,” which means that they isomerize to thecis form under the time, temperature, and/or pH conditions of the priorart. Furthermore, the proper balance between time, temperature, pH, andborohydride equivalents must achieve essentially complete reduction ofthe IA if the DHIA is to be light stable, and yet not epimerize thetrans isomer to the cis one, or form undesirable and haze formingover-reduced products. The following rules can be applied to guide oneskilled in the art:

(1) the lower the pH, the longer the reaction time.

(2) the higher the pH, the easier to eliminate residual IA.

(3) the higher the pH the more degradation products, especially in thehumulinic acid classification.

(4) the longer the reaction time, the more complete the reduction andthe more by-products formed.

(5) the more molar equivalents (ME) of borohydride, the faster thereduction, accompanied by an increase in by-products.

(6) The lower the pH, the more rapidly the borohydride decomposes withthe evolution of nascent hydrogen, which can also create undesirableby-products.

The reactions were studied for optimization of the conditions, sinceprior art conditions did not produce the inventive products . Marginallyacceptable DHIA in acceptable yield can be made at a pH of up to about11.8. Above that pH, formation of the cis isomer predominates,especially above 50° C. Increasing the mole equivalents of borohydridebeyond 0.75 moles increases by-product formation to above 15%. Byreducing the pH to 11, and the time to two hours, little epimerizationoccurs and by-product formation is about the same as at pH 11.7. Byreducing the pH to 10.5 , and the temperature to 50° C., epimerizationbecomes negligible but the time required to achieve about the sameresidual IA is 5 to 7 hours. (Shorter reaction times leave unreduced IA,which must be below 0.5% if the product is to be light stable).

Table 16-1 compares the prior art ranges with feasible and optimalconditions for reduction of IA and THIA. It will be noticed that thecombination of preferred conditions do not coincide with the prior art.For HHIA, which epimerizes more slowly than DHIA, it is noticed that thepH can safely be raised above the pH for DHIA

TABLE 16-1 Comparative Reaction Conditions with Prior- art. time, conc,% pH temp, ° C. ME, BH4 hrs DHIA range 2-28 10-11.8 25-75 0.4-0.81 3-6preferred 8-15 10-11 45-65 0.5-0.7 3-5 Gold.- 17 12.7 79 0.5 2 640Gold.- 5 12 60-65 0.75 3 810 West.-326 13 13.5 60 0.63 3 Koch -879 1 1125 1.97 4 HHIA range 4-25 10-12.2 25-80 0.4-0.81 3-6 preferred 7-1310.5— 45-65 .5-.7 3-5 11. 9 Todd-731 5 13.5 70 0.32 3 Worden 1 11.1 251.84 4

The procedures of both Koch and Worden, who first disclosed DHIA andHHIA, were repeated. It should be noticed that both Koch and Worden use7.9 to 8.5 times the theoretical hydrogen equivalents of borohydride,whereas the more recent prior art uses a maximum of about 3 times, andthe preferred range for making the claimed products is about 1.6 to 2.8hydrogen equivalents. (Borohydride contains four active hydrogen atomsper mole, and only one mole of hydrogen is used up in reduction, so thetheoretical requirement of borohydride is 0.25 molar equivalents). Thegreat excess of hydrogen equivalents produced very high levels ofover-reduced and other by products when their examples were repeated. Asa consequence, the prior art as taught by them must be consideredobsolete in view of Westermann and Todd.

Both the Koch and Worden products formed gummy precipitates atconcentrations of 10 to 20% in alkaline water, and turbid solutions at1%.

Byrne and Shaw (J. Chem. Soc.,(C), 2810-2813, 1971) found that fourhydrogen equivalents (1 molar equivalent) of borohydride reducedcis-THIA, whereas eight hydrogen equivalents (2 molar equivalents) wereneeded to reduce trans-THIA. Because of this large excess, as in thecase of Koch and Worden, substantial amounts of over-reduced and otherby-products must have been formed. They did not show solid or liquidmixtures of cis and trans isomers.

An HPLC analysis which displays the spectra of the individual peaks, ifthey are clearly separated, permits the measurement of the over-reducedproducts made by the prior art and the substantial absence of theseproducts made when the lower hydrogen equivalents employed in theprocess described in this specification are used, and as claimed.

It is to be understood that the present invention is not to be limitedto the exact details of operation, or to the exact compounds,compositions, methods, procedures, or embodiments shown and described,as various modifications and equivalents will be apparent to one skilledin the art, wherefore the present invention is to be limited only by thefull scope which can be legally accorded to the appended claims.

We claim:
 1. The process of reducing (a) isoalpha acids (IA) to produce dihydroisoalpha acids (DHIA) or (b) tetrahydroisoalpha acids (THIA) to produce hexahydroisoalpha acids (HHIA), the DHIA or the HHIA product having a trans to cis isomer ratio greater than 10%, the reduction being carried out in an aqueous medium at a pH of about 8.5 to about 12.4 using a borohydride.
 2. The process of claim 1 wherein IA are reduced to DHIA having a trans to cis isomer ratio greater than 10% using less than about 0.81 molar equivalents of a borohydride and a pH up to about 11.8.
 3. The process of claim 1 wherein THIA are reduced to HHIA having a trans to cis isomer ratio greater than 10% using less than about 0.81 molar equivalents of a borohydride.
 4. The process of claim 1 in which the temperature at which the reduction is carried out is up to about 75° C. and in which the reaction is terminated before the trans to cis isomer ratio of the product DHIA or HHIA becomes less than 10%.
 5. The process of claim 1 wherein the reduction is carried out with up to about 0.65 molar equivalents of borohydride.
 6. The process of claim 5 wherein the reduction is carried out with up to about 0.55 molar equivalents of borohydride.
 7. The process of claim 1 in which a lower alkanol is also present.
 8. The process of claim 1 wherein the pH of the aqueous medium is buffered at about 12.4 or below.
 9. The process of claim 8 wherein the buffering agent is selected from potassium and sodium salts of phosphates, citrates, and borates.
 10. The process of claim 1 in which a non-reactive water-immiscible solvent is also present.
 11. The process of claim 10 in which the water-immiscible solvent is a hydrocarbon containing 10 or less carbon atoms.
 12. The process of claim 1 in which hydrocarbon-soluble haze-forming substances are removed from the DHIA or HHIA product by admixing a hydrocarbon with the aqueous DHIA or HHIA phase and removing the hydrocarbon phase, wherein the aqueous DHIA or HHIA phase is 15% or less DHIA or HHIA, and wherein the pH is up to about 10.5, to give a DHIA or HHIA product wherein the remaining hydrocarbon-soluble substances are less than 3% by weight of the DHIA or HHIA product.
 13. The process of claim 12 in which the pH is about 7.5-9.5.
 14. The process of claim 13 wherein the hydrocarbon has 6 to 10 carbon atoms.
 15. The process of claim 1 in which the final aqueous DHIA or HHIA phase is concentrated at a pH below about 10.5 and greater than 6 by evaporation of water, to give a concentrated aqueous phase containing between about 5% and about 40% DHIA or HHIA.
 16. The process of claim 15 wherein the pH is between 6.5 and 8.5 and the DHIA or HHIA concentration is less than about 25%.
 17. The process of claim 1 wherein the borohydride is selected from the group consisting of sodium borohydride and potassium borohydride.
 18. The process of claim 2 wherein the DHIA having a trans to cis isomer ratio greater than 10% is subsequently converted to HHIA having a trans to cis isomer ratio greater than 10% by catalytic hydrogenation. 